Cloning And Functional Analyses Of AmGPAT And AmFAD8 Genes From Ammopiptanthus Mongloicus

Thylakoid in chloroplast is the site of photoreaction in photosynthesis of plants, and it is also one of the most sensitive parts to temperature. PG (phosphatidylglycerol) is the unique phospholipid in thylakoid membranes, and the unsaturation degree of its fatty acids is closely related to the sensitivity of plants to low temperature. Both the chloroplastic GPAT (sn-glycerol-3-phosphate acyltransferase) and FADs (fatty acid desaturases) are key factors that determine the content of unsaturated fatty acids in PG. In this study, we cloned the cDNAs of AmGPAT and AmFAD8 genes from Ammopiptanthus mongolicus, a strong cold-resistant plant. Then, we primarily analyzed the functions of AmGPAT in response to and resisting temperature stress. The main results are as follows:(1) The complete coding region cDNA of AmGPAT was cloned by RT-PCR. It was consist of 1383bp encoding a protein of 460 aa residues. Sequence analysis revealed that it was to be hydrophilous protein,with N-chloroplast transit peptide. It is part of soluble protein in chloroplast.(2) Semi-quantitative RT-PCR indicated the higher expression of AmGPAT in normal condition and up-regulation of the gene by low temperature stress in the chamber and nature; The expression was suppressed overall under high temperature and was slightly affected under drought stress.(3) The cDNA fragment of AmGPAT was successfully ligased to plant over-expression vector. After it was transformed into Arabidopsis, the stress resistance analysis of the homozygous lines of T3 generation was carried out. The results suggested that the survival rate was obviously higher in transgenic lines, compared to WT seedings, and the growth in the recover process was better than WT under freezing injury. In addition, the maximal photochemical efficiency of PS Ⅱ (Fv/Fm) in the transgenic lines recovered faster than in WT after chilling stress. But the high temperature and drought resistances in the transgenic lines were lower than in the WT. These results above demonstrated that the functions of AmGPAT mainly responded and resisted to low-temperature stress.(4) The complete coding region cDNA of AmFAD8 was cloned by RT-PCR from Ammopiptanthus mongolicus. It is consist of 1526bp encoding a protein of 457 aa residues. The cDNA fragment of AmFAD8 was successfully ligased to plant over-expression vector. Then it was transformed into Arabidopsis and obtained the T1 generation.